The last three decades has seen considerable effort in the development of improved methods for the isolation and purification of nucleic acids and proteins from biological sources. This has been due mainly to the increasing applications of these materials in the medical and biological sciences. Although it is advantageous in many occasions to isolate nucleic acids and proteins from the same sample, effective methods have been lacking.
Currently the most used protocol for simultaneous isolation of genomic DNA, RNA and proteins from different types of samples is a solution phase based method which utilizes TRI reagent (TRIzol; BioTechniques, 35, 450-456 (2003)). This process is based on organic/aqueous extractions and it involves the use of toxic chemicals, which include chloroform, bromochloropropane, phenol and guanidine isothiocyanate. The other drawback of this solution phase method is the difficulty in separation of the layers. If the layers are not completely separated there will be considerable amount of cross contamination.
Recently Norgen Biotek launched a silicon carbide bead based chromatography product for the isolation of DNA, RNA and protein (RNA/DNA/Protein Purification Kit). Macherey-Nagel offers a product for isolation of RNA and Protein (NUCLEOSPIN® RNA/Protein Kit). This product can be combined with a buffer set commercialized by Macherey-Nagel to accomplish the isolation of all three components. All these products are silica based technologies.
Another method for the simultaneous isolation of genomic DNA, RNA and proteins is recently disclosed in PCT application WO 03/062462 by Dynal Biotech. In this patent application, the authors propose using distinct solid supports for the isolation of DNA, mRNA and protein. The specific solid supports are dT or dU oligo derivatized support, carboxylic acid derivatized support, dynabeads derivatized with amine and magnetic particles. In summary they use different types of resins for the isolation of three different components from one sample. This is done by a stepwise addition of a specific solid support to the lysate, removal of this support and then the addition of a second support to the lysate to isolate the second component and so on. Here DNA is isolated by complexation mechanism and mRNA is isolated by affinity methods.
Currently several procedures are available for the chromatographic purification of DNA (genomic and plasmid) and RNA, for example, by employing silica based membrane purification, size exclusion chromatography, reversed phase chromatography, gel filtration, magnetic bead based purification, or ion-exchange chromatography. Ion exchange chromatography is one of the most commonly used separation and purification methods and has been used for purification of plasmid DNA, genomic DNA and RNA. See for example, U.S. Pat. No. 6,410,274 (Bhikhabhai), U.S. Pat. No. 6,310,199 (Smith et al), U.S. Pat. No. 6,090,288 (Berlund et al), U.S. Pat. No. 5,990,301 (Colpan et al), U.S. Pat. No. 5,856,192, U.S. Pat. No. 5,866,428 (Bloch), U.S. Pat. No. 5,801,237 (Johansson), EP 1125943 B1 (Macherey-Nagel GmbH & Co), EP 992583 B1, EP 616639 (Qiagen), U.S. Pat. No. 5,707,812 and U.S. Pat. No. 5,561,064 (Vical Inc.).
While anion exchange chromatographic procedures for the purification of nucleic acids have been extensively referenced, one of the shortcomings of current protocols is the impaired recovery of nucleic acid during the elution step. Addition of organic agents such as polyols and alcohols during adsorption and desorption has been shown to improve selectivity and recovery during anion exchange purification of DNA (Tseng, W. C. et al, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., (2003), 791(1-2), 263-72). Recently, improvements have been made to the elution buffer that specifically address the recovery issues often seen during DNA desorption from anion exchange resins (See U.S. Pat. Nos. 7,655,792 and 7,655,793). These improved methods whether by an increase in the pH of the elution solution, or by the inclusion of specific salt compositions, greatly increase the recovery rate of bound nucleic acids from an anion exchange column.
There remains a need for improved methods for the simultaneous isolation of genomic DNA, RNA and proteins from a single sample.